The goal of this project is to elucidate the effect of pre-existing adeno-associated virus-2-specific T cell-mediated immunity on hepatic rAAV vector-mediated gene transfer. Recombinant (r) AAV-2 vectors have yielded promising results in gene replacement therapy of immunocornpetent experimental animals suggesting that they are comparatively poorly immunogenic. Humans are generally exposed to AAV-2 antigens during childhood through infection by AAV-2 accompanying another virus, a so-called helper virus, such as an adenovirus or a herpes virus. Under circumstances where the poorly immunogenic AAV-2 is introduced with a helper virus that causes a strong inflammatory reaction, the helper virus is expected to provide an adjuvant effect that should facilitate the induction of a strong adaptive immune response to antigens of AAV-2. Human subjects unlike experimental rodents or canines are thus likely to have antigen-experienced T cells to AAV-2 that may become activated more readily upon rAAV-2-mediated gene transfer than has been appreciated by pre-clinical studies conducted thus far. Potentially confounding this problem is that AAV-2 persists in humans in a form that suggests that its genome may continue to be transcribed. Even very low levels of persistent AAV-2 antigen would maintain the immune system at a heightened stage of activation. Pre-existing T cell-mediated immunity to AAV-2 may also affect the use of simian AAVs for gene replacement therapy due to shared T cell epitopes between human and simian origin AAVs. One of the challenges of this application is to mimic natural AAV infection of humans in an experimental small animal species suited to conduct an in depth study of immune responses. For this, inbred mice will be used although they are not natural hosts for AAV-2 or its natural helper viruses. To overcome this limitation we developed and will continue to refine in aim 1 an experimental system to induce AAV capsid-specific T cell responses in mice, which resemble functionally those in human subjects. In aim 2, we will characterize the immune response to AAV-2 capsid in depth using the most suitable mouse model. In aim 3, we will use the model to test the effect of pre-existing immunity to AAV-2 on the efficacy, immunogenicity and longevity of rAAV-2-mediated gene transfer. Experiments will be extended in aim 4 to a vector system based on simianderived AAV-8 to assess if a heterologous rAAV vector can overcome pre-existing immunity to AAV-2.